- No binding
- High background signal
- Target protein not present in the cell extract, lost or destroyed during first step of preparation.
Check that the protein of interest is actually present in the cell extract/lysate. Prepare fresh extracts, do not freeze them. Use suitable protease inhibitors.
- Washes are too strong.
Decrease the amount of washing steps as well as concentration of the detergent and salt.
- Large number of competing proteins are present in the sample.
Centrifugate the cell extract/lysate at 10 000 x g for 30 minutes before adding the antibody. This should help to remove insoluble aggregates.
- Interfering substances present in the cell extract/lysate.
Make sure that no DTT, mercaptoethanol or other reducing agents are added to your lysates. They will be the reason for antibody breakdown. Extreme pH values and increased detergent concentrations might also interfere with establishing of the antigen-antibody complex.
- Wrong beads used to isolate the antigen-antibody complex.
Check which Protein A and G beads will bind your class of antibody the best.
- Antibody not able to perform in immunoprecipitation or used in too low amount.
A precipitating antibody must be changed to another one. Usually polyclonal antibodies are better in immunoprecipitation assays than monoclonal antibodies. The amount of antibody used in this assay must be titrated. In some cases, if for instance antibodies were raised against denatured antigen, native epitopes might not be recognized.
- Is your target protein glycosylated? Glycosylation can contribute to steric hindrance of the epitope and unable the antibody to reach it, by simply masking it. You can try to denature the protein by heating up the whole extract for several minutes. However, this will also contribute to aggregate formation of other proteins present in the extract.
High background signal
- Antibody concentration used is too high.
Titrate the antibody used.
- Non-specific binding of other proteins either to the Protein A or G beads or to the antibodies.
Worth considering in immunoprecipitation:
- SeizeTM X Immunoprecipitation Kits (Pierce)
- Dynal beads (Dynal)
Both will allow coupling of the primary antibody permanently and bypass the problem with light or heavy immunoglobulin chains interfering with the detection of a target protein, following gel electrophoresis.